The present invention is directed to nucleic acid molecules encoding vespid venom allergens, in particular venom enzymes such as phospholipase and hyaluronidase, or fragments thereof, recombinant vectors comprising such nucleic acid molecules, and host cells containing the recombinant vectors. The invention is further directed to expression of such nucleic acid molecules to produce a recombinant vespid venom enzyme, such as phospholipase or hyaluronidase, or recombinant fragments thereof. Such an allergen and fragments thereof are useful for diagnosis of allergy, for therapeutic treatment of allergy, for the treatment of immune system related diseases or disorders, or symptoms related thereto, and for the modulation of immune response towards an immunogen.
Insect sting allergy to bees and vespids is of common occurrence. The vespids include hornets, yellowjackets and wasps (Golden, et al., 1989, Am. Med. Assoc. 262:240). Susceptible people can be sensitized on exposure to minute amounts of venom proteins; as little as 2-10 xcexcg of protein is injected into the skin on a single sting by a vespid (Hoffman and Jacobson, 1984, Ann. Allergy. 52:276).
There are many species of hornets (genus Dolichovespula), yellowjackets (genus Vespula) and wasp (genus Polistes) in North America (Akre, et al., 1980, xe2x80x9cYellowjackets of America North of Mexico,xe2x80x9d Agriculture Handbook No. 552, U.S. Department of Agriculture). The vespids have similar venom compositions (King, et al., 1978, Biochemistry 17:5165; King, et al., 1983, Mol. Immunol. 20:297; King, et al., 1984, Arch. Biochem. Biophys. 230:1; King, et al., 1985, J. Allergy and Clin. Immunol. 75:621; King, 1987, J. Allergy Clin. Immunol. 79:113; Hoffman, 1985, J. Allergy and Clin. Immunol. 75:611). Their venom each contains three major venom allergens, phospholipase (37 kD), hyaluronidase (43 kD) and antigen 5 (23 kD) of as yet unknown biologic function.
In addition to the insect venom allergens described above, the complete amino acid sequence of several major allergens from different grass (Perez, et al., 1990, J. Biol. Chem. 265:16210; Ansari, et al., 1989, Biochemistry 26:8665; Silvanovich, et al., 1991, J. Biol. Chem. 266:1204), tree pollen (Breiteneder, 1989, EMBO J. 8:1935; Valenta, et al., 1991, Science, 253:557), weed pollen (Rafnar, et al., 1991, J. Biol. Chem. 266:1229; Griffith, et al., 1991, Int. Arch. Allergy Appl. Immunol. 96:296), mites (Chua, et al., 1988, J. Exp. Med. 167:175), cat dander (Griffith, et al., 1992, Gene. 113:263), and mold (Aruda, et al., 1990, J. Exp. Med. 172:1529; Han, et al., 1991, J. Allergy Clin. Immunol. 87:327) have been reported in the past few years. These major allergens are proteins of 10-40 kD and they have widely different biological functions. Nearly all allergens of known sequences have a varying extent of sequence similarity with other proteins in our environment.
Antibody responses to proteins require the collaboration of T helper and B lymphocytes and antigen presenting cells (APC). The antigen receptors of B cells are the membrane-bound antibody (Ab) molecules, which recognize and bind immunogens directly. The antigen receptors of T cells (TCR) only recognize and bind complexes of antigenic peptide-MHC class II molecule. Immunogens are first processed by APC into peptides that are presented on the surface of APC in association with the MHC class II molecules (Unanue, 1992, Current Opinion in Immunol 4:63). As MHC molecules are highly polymorphic in individuals, they have different specificity of binding antigenic peptides (Rothbard and Gefter, 1991, Ann. Rev. Immunol. 9:527). This is one mechanism for genetic control of immune response.
T helper cells are activated when the antigen receptor binds the peptide-MHC complex on the surface of APC. Activated T cells secrete lymphokines. In mice (Street and Mosmann, 1991, FASEB J. 5:171) and apparently in humans (Wierenga, et al., 1990, J. Immunol. 144:4651; Parronchi, et al., 1991, Proc. Natl. Acad. Sci. USA. 88:4538) the T helper cells can be divided into different types on the basis of their patterns of lymphokine production. Primarily, T helper cells divide into two groups: TH1 cells producing IL-2 and IFN-xcex3, and TH2 cells producing IL-4 and IL-5. These lymphokines in turn influence the antigen-activated B cells to differentiate and proliferate into plasma cells secreting Abs of different isotypes. IL-4 is one lymphokine known to influence IgE synthesis (Finkelman, et al., 1990, Ann. Rev. Immunol. 8:303).
It is believed that the entire accessible surface of a protein molecule can be recognized as epitopes by the antigen receptors of B cells, although all epitopes are not necessarily recognized with equal likelihood (Benjamin, et al., 1984, Ann. Rev. Immunol. 2:67). B cell epitopes of a protein are of two types: topographic and linear. The topographic type consists of amino acid residues which are spatially adjacent but may or may not be sequentially adjacent. The linear type consists of only sequentially adjacent residues. X-ray crystallographic data of Agxe2x80x94Ab complexes indicate the size of their complementary binding region to have 16-17 amino acid residues (Amit, et al., 1986, Science 233:747). Phospholipase, like other protein antigens, can have both types of B cell epitopes or only one. For example, vespid antigen 5s have both types. Bee venom melittin appears to have only one B cell epitope of linear type (King, et al., 1984, J. Immunol. 133:2668).
T cell epitopes of proteins consist of only the linear type since they are peptides that have been processed in the lysosomes of APC by proteases of unknown specificity (Unanue, 1992, Curr. Op. Immunol. 4:63). Analysis of naturally processed antigenic peptides bound to MHC class II molecules indicates that their size ranges from about 13 to 17 amino acid residues, but analysis of synthetic peptide-MHC class II molecule complex for their T cell proliferate response suggests a minimal size of about 8 amino acid residues (Cf. Rudensky et al., 1991, Nature 353:622). Studies suggest that T cell epitopes are distributed throughout the entire protein molecule, and they may function as major or minor determinants depending on the MHC haplotype of the immunized host (Roy, et al., Science 244:572; Gammon, et al., 1987, Immunol. Rev. 98:53; O""Hehir et al., 1991, Ann. Rev. Immunol. 9:67).
Hypersensitivity of the immediate type is known to be caused by the presence of allergen-specific IgE. IgE is found in the circulation and bound to specific IgE-Fc receptors on mast cells and basophils. Cross-linking of cell-bound IgE by allergens leads to release of histamine, leukotrienes and other chemical mediators that cause the allergic symptoms. IgE is one of the different isotypes of immunoglobulins. As pointed out above, lymphokines secreted by T cells influence isotype switch events in B cells.
Because of the central role of TH2 cells in determining the isotypes switch event of B cells, the T cell epitopes of several allergens have been mapped (Cf. O""Hehir et al., supra). These allergens include ragweed Amb xcex1 III, rye grass Lol p I, cat Fel d I, mouse urine Mus m I, midge Chi t I, bee venom phospholipase A2 (Dhillon, et al., 1992, J. Allergy Clin. Immunol. 90:42) and melittin (Fehlner, et al., 1991, J. Immunol. 146:799). The data do not reveal any unusual or common structural features. However, any conclusion from these data is qualified as these data are collected from humans and mice of different haplotypes.
Normally hosts are tolerant to the dominant B and T cell epitopes of self proteins by clonal deletion and anergy. However this tolerance can be broken under certain circumstances (Gammon, et al., 1991, Immunol. Today. 12:193; Basten, et al., 1991, Immunol. Rev. 122:5). It has been suggested that self-tolerance is broken in autoimmune diseases through encounters with foreign proteins that are similar to host proteins. Therefore the sequence similarity of allergens with autologous proteins is of interest for closer investigation.
Mature B cells are activated in response to multi-valent antigens which can cross-link cell surface Ig receptors (DeFranco, 1987, Ann. Rev. Cell Biol. 3:143), and they are rendered anergic in response to mono-valent antigen (Basten, et al., 1991, supra). Antigen activation of T cells requires not only the integration of TCR with peptide-MHC complex but also with other co-stimulating signals on the surface of APC (Schwartz, 1990, Science 248:1349; Jenkins and Miller, 1992, FASEB J. 6:2428). Interaction of TCR with peptide-MHC complex in absence of co-stimulating signals can lead to T cell anergy.
The molecular mechanism of B or T cell anergy is not yet understood (Cf. Schwartz, 1990, supra; Jenkins and Miller, 1992, supra; Ales-Martinez, et al., 1991, Immunol. Today 12:201). In vitro studies with T cell clones reveals that occupancy of TCR by artificial peptide-MHC complex in absence of co-stimulating signals leads to altered intracellular signal transduction and/or repressor gene activation which can prevent lymphokine transcription.
Early studies have shown that the physical state of the immunogen and the route of immunization are important variables in determining the outcome of an immune response. In the light of our current understanding, these variables may well influence antigen presentation so as to have T and B cell activation or anergy.
One way to treat allergic diseases is by immunotherapy which involves repeated subcutaneous injections of the offending allergen(s) into patients. The amounts of allergens which can be injected are limited by the danger of unwanted systemic allergic reaction in patients. For most patients following immunotherapy, their allergen-specific IgE levels initially rise followed with gradual decrease of their allergen-specific IgG levels, and there is also downregulation of allergen-specific T cell responses (P. S. Norman, 1993, Current Op. Immunol. 5:968).
Because of the undesirable systemic reaction on immunotherapy with native allergens, there has been continued interest in the development of modified allergens with reduced allergenic activities for immunotherapy (T. P. King, 1993, in xe2x80x9cBronchial Asthma,xe2x80x9d edited by E. B. Weiss and M. Stein, Little Brown, Boston, pp. 43-49; R. E. O""Hehir et al., 1991, supra).
Two reports have appeared recently on the use of T cell epitope peptides to modulate allergen-specific immune responses. One report is on the subcutaneous injection of mice with two peptides from the major cat allergen Fel d I to decrease T cell response to the entire molecule Fel d I (Briner et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7608-12). Another is on the intranasal therapy with a peptide from the major mice allergen Der p I to suppress allergen-specific response in naive or sensitized mice (Hoyne et al., 1993, J. Exp. Med. 178:1783-1788).
Since an MHC class II molecule of any one haplotype can bind a wide range of peptides in its binding groove, it may be possible to modulate T cell response by inhibition of allergen-derived T cell epitope binding to MHC molecules with other peptides. For example, a mouse lysozyme peptide which is not immunogenic by itself in H-2k mice inhibits T cell response to hen egg white lysozyme (Adorini and Nagy, 1990, Immunol. Today. 11:21). Another example is the in vitro inhibition of T cell response to a mite allergen by an influenza HA peptide (O""Hehir et al., 1991, J. Allergy Clin. Immunol. 87:1120).
Experimental autoimmune encephalomyelitis (EAE) in mice or rats is a well-studied model for multiple sclerosis. Many studies have identified immunodominant T cell determinants for myelin basic protein, which is used to induce this condition. Peptides that correspond to immunodominant epitopes of myelin basic protein can induce tolerance to the same peptide antigen or to the intact myelin basic protein. The same peptides that induced tolerance could also induce T cell anergy in an ongoing autoimmune response (Gaur et al., 1992, Science 259:1491-1494).
Immune response to an immunogen/allergen depends in part on the genetic make-up of the host, the route and mode of immunization and the immunogen/allergen. The extent to which a vespid venom allergen determines the outcome of IgE response is not known. How many B and T cell epitopes does each vespid venom allergen have? Are there immunodominant B or T cell epitopes of a vespid venom allergen recognized by different or all susceptible individuals? Are there T cell epitopes which favor IgE class switch events in B cells? Does antigenic cross reactivity of vespid venom allergens with host proteins play a role as to why some proteins are more allergenic than others are? Can tolerance to a multi-valent vespid venom allergen be induced by treatment with a single or a combination of B or T cell epitopes?
Thus, there is a need in the art to delineate the B and helper T cell epitopes of major vespid venom allergens. There is a particular need to delineate the B and helper T cell epitopes of the vespids hornet (e.g., Dolichovespula arenaria), yellowjacket (e.g., Vespula vulgaris) and wasp (e.g., Polistes annularis). In particular, the major vespid venom allergens phospholipase and hyaluronidase are appropriate targets for determining the important B and T cell epitopes. In order to fully address the basis for allergic response to vespid allergens, and to develop allergen-based immunotherapies, the cDNA and protein sequences of several homologous allergens need to be investigated. Moreover, vectors suitable for high level expression in bacteria and eukaryotic cells of vespid allergens or their fragments should be developed. Recombinant vespid allergens and their fragments may then be used to map their B and T cell epitopes in the murine and, more importantly, human systems by antibody binding and T cell proliferation tests, respectively.
There is a further need to determine whether there is cross reaction of the T and B cell epitopes of vespid allergens with other environmental and/or autologous proteins. Thus there is a need to determine whether vespid allergens share partial identity with other environmental proteins, especially with autologous proteins, and more importantly, to obtain the sequences of the regions of the partial identity, in particular the specific amino acid sequences of such regions of partial identity. There is a further need to determine the level of cross reactivity of vespid allergens with other proteins at the B cell and T cell level, the relevance of this cross reactivity, and whether such cross reactivity is pathological, i.e., involved in or responsible for allergy, or beneficial, i.e., inhibitory of allergy.
There is also a need in the art to use peptides having T or B cell epitopes of vespid venom allergens to study induction of tolerance in mice and induction of tolerance in humans.
There is a further need to test whether a modified peptide inhibits allergen T cell epitope binding to MHC class II molecule, or induces T cell anergy, or both.
Thus, there is a need in the art for the sequence information about vespid venom allergens, and a plentiful source of such allergens for immunological investigations and for immunological therapy of the allergy.
Furthermore, due to the overuse of antibiotics throughout the world, and to the spread of numerous viruses, such as HIV, Ebolla, etc., efforts have been made to produce new xe2x80x9csuperxe2x80x9d antibiotic medication, and compounds which have activity against viruses. For example, AZT has been developed, along with protease inhibitors to treat subjects suffering from HIV. However, the costs of developing new xe2x80x9csuperxe2x80x9d antibiotics and anti-viral medications are enormous.
Hence, what is needed are agents, and pharmaceutical compositions for treating immune system related diseases or disorders whose activity is not dependent necessarily on combating the particular virus or pathogen, but rather modulate or potentiate the immune system ability to combat the disease or disorder, thereby ameliorating the disease or disorder, or a symptom related thereto.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
The present invention provides nucleic acid molecules encoding vespid venom enzymes, immunomodulatory fragments thereof, or derivatives or analogs thereof. In particular, the invention is directed to nucleic acid molecules encoding a vespid venom phospholipase, and a vespid venom hyaluronidase, In specific embodiments, a nucleic acid molecule of the invention encodes an immunomodulatory portion of a T cell epitope of a vespid venom enzyme. In another embodiment, a nucleic acid molecule of the invention encodes an antigenic portion of a B cell epitope of a vespid venom enzyme
Hence broadly, the present invention extends to an isolated nucleic acid molecule encoding a vespid venom enzyme, conserved variant thereof, immunomodulatory fragment thereof, or derivative, or analog thereof. Examples of vespid venom enzymes which can be encoded by an isolated nucleic acid molecule of the invention include, but are not limited to phospholipase and hyaluronidase. Moreover, enzymes, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, from the venom of numerous vespid venoms can be encoded by an isolated nucleic acid molecule of the invention. A particular example comprises vespids of the genus Polistes, and particularly the species annularis. Hence, in a particular embodiment, the present invention extends to an isolated nucleic acid molecule encoding a phospholipase A1, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, from the genus Polistes and the species annularis, wherein the isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Moreover, the present invention extends to an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule which encodes a vespid venom enzyme, conserved variants thereof, immunomodulatory fragments thereof, derivatives, or analogs thereof. Examples of such enzymes include, but are not limited to phospholipase and hyaluronidase. Furthermore, examples of vespids having applications herein include those of the genus Polistes and particularly of the species annularis. Hence in a particular embodiment, the present invention extends to an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, which encodes phospholipase A1 comprising an amino acid sequence of SEQ ID NO:64, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof.
Another example of a vespid venom enzyme which can be encoded by an isolated nucleic acid molecule of the invention comprises hyaluronidase. Hence, the present invention extends to an isolated nucleic acid molecule, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, which encodes vespid venom hyaluronidase, conserved variants thereof, fragments thereof, or analogs or derivatives thereof. An isolated nucleic acid molecule of the invention encoding vespid venom hyaluronidase can be obtained from numerous vespids. Examples include, but are not limited to, vespids of the genus Polistes, and particularly, the species annularis. Hence, in a particular embodiment, the present invention extends to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:67, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, which encodes hyaluronidase from Polistes annularis comprising an amino acid sequence of SEQ ID NO: 68, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof.
Moreover, the present invention extends to an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:67, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof.
Moreover, the present invention further extends to an isolated nucleic acid molecule encoding a vespid venom enzyme, or an immunomodulatory fragment, derivative or analog thereof, wherein the isolated nucleic acid molecule encodes an immunomodulatory portion of a T cell epitope of the vespid venom enzyme. Likewise, the present invention extends to an isolated polypeptide comprising an immunomodulatory portion of a T cell epitope of a vespid venom enzyme, wherein the polypeptide is encoded by an isolated nucleic acid molecule of the invention. Examples of vespid venom enzymes for which isolated nucleic acid molecules of the present invention encode an immunomodulatory portion of a T cell epitope include, but certainly are not limited to, phospholipase and haluronidase. Furthermore, the phospholipase A1 and haluronidase may originate from numerous vespid venoms, including from vespids the genus Polistes, and particularly from the species annularis. 
What""s more, the present invention extends to an isolated nucleic acid molecule encoding a vespid venom enzyme, or an immunomodulatory fragment, derivative, or analog thereof, wherein the isolated nucleic acid molecule encodes an antigenic portion of a B cell epitope of the vespid venom enzyme. Examples of such vespid venom enzymes include phospholipase, and hyaluronidase which can originate from numerous vespids, including from the genus Polistes, and particularly, from the species annularis. 
Furthermore, the present invention extends to an isolated polypeptide comprising an antigenic portion of a B cell epitope of a vespid venom enzyme, wherein the polypeptide is encoded by an isolated nucleic acid molecule of the invention. Examples of vespid venom enzymes for which isolated nucleic acid molecules of the present invention encode an antigen portion of a B cell epitope include phospholipase and hyaluronidase. Furthermore, the phospholipase and hyaluronidase may originate from numerous vespid venoms, including from vespids the genus Polistes and particularly the species annularis. 
The present invention further extends to an isolated expression vector comprising isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, operatively associated with a promoter. Likewise, the present invention extends to an isolated expression vector comprising an isolated nucleic acid molecule which is hybridizable to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. Numerous promoters commercially available to the skilled artisan can be used in this aspect of the invention. Examples include, but are not limited to immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus, late promoters of vaccinia, late promoters of polyoma, the lac the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast xcex1 mating factor, to name only a few. Numerous examples of expression vectors having applications herein, and which are also readily available to the skilled artisan are described infra.
Moreover, the present invention extends to an isolated expression vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:67, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, operationally associated with a promoter. In addition, the present invention extends to an isolated expression vector comprising an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:67, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof. Examples of expression vectors and promoters having applications in this aspect of the invention are described infra.
The present invention further extends to a method for producing a vespid venom enzyme, such as phospholipase, conserved variants thereof, fragments thereof, or analogs or derivatives thereof by expression an isolated nucleic acid molecule of the invention. Such production provides a plentiful source of the vespid enzymes for diagnosis and therapy. An example of such a method of the invention for producing vespid venom phospholipase comprises the steps of:
(a) culturing a host cell transformed or transfected with an expression vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, operationally associated with a promoter, so that the host cell produces the vespid venom phospholipase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof; and
(b) recovering the vespid venom phospholipase, conserved variants thereof, immunomodulatory fragment thereof, or analogs or derivatives thereof produced from the culture, the host cell, or both.
Another method of producing a vespid venom phospholipase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof, which is encompassed by the present invention, comprises:
(a) culturing a host cell transformed with an expression vector comprising an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the isolated nucleic acid molecule is operationally associated with a promoter, so that the vespid venom phospholipase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof, is produced by the host cell; and
(b) recovering the vespid venom phospholipase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof so produced from the culture, the host cell, or both.
In a particular example, the methods set forth above have ready applications in producing phospholipase A1 from vespids of the genus Polistes, and particularly from the species annularis, wherein the phospholipase A1 comprises an amino acid sequence of SEQ ID NO:67, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof.
Moreover the present invention extends to methods for producing vespid venom hyaluronidase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof. An example of such a method comprises:
(a) culturing a host cell transformed with an expression vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:67, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the isolated nucleic acid molecule is operationally associated with a promoter, so that the vespid venom hyaluronidase, conserved variant thereof, immunomodulatory fragment thereof, analog or derivative thereof, is produced by the host cell; and
(b) recovering the vespid venom hyaluronidase, conserved variant thereof, fragment thereof, or analog or derivative thereof, from the culture, the host cell, or both.
Another method for producing a vespid venom hyaluronidase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, comprises the steps of:
(a) culturing a host cell transformed with an expression vector comprising an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:67, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the isolated nucleic acid molecule is operationally associated with a promoter, so that the vespid venom hyaluronidase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof is produced by the host cell; and
(b) recovering the vespid venom hyaluronidase, conserved variant thereof, immunomodulatory fragment thereof, or analog or derivative thereof so produced, from the culture, the host cell, or both.
Hence, immunomodulatory fragments or antigenic portions of a B cell epitope of vespid venom hyaluronidase, particularly Polistes annularis venom hyaluronidase, can be produced with the present invention. These fragments can be used therapeutically for the treatment of vespid venom enzyme-specific allergic conditions, to modulate immune response towards an immunogen, or to treat an immune system related disease or disorder, or a symptom related thereof. Furthermore, the therapeutic treatment can be highly specific and individualized, since the invention allows production of a vespid venom enzyme polypeptide that has immunomodulatory activity in any individual or group of individuals.
The present invention further extends to pharmaceutical compositions effective for the treatment of a vespid venom allergen-specific allergic condition. In particular, the present invention extends to a pharmaceutical composition comprising a polypeptide encoded by an isolated nucleic acid molecule which encodes an immunomodulatory portion of a T cell epitope of a vespid venom enzyme, and a pharmaceutically acceptable carrier thereof. Another pharmaceutical composition of the invention comprises a polypeptide encoded by an isolated nucleic acid molecule which encodes a vespid venom enzyme, wherein the polypeptide comprises an antigen portion of a B cell epitope of a vespid venom enzyme. In a particular embodiment of the invention, the pharmaceutical composition comprises a polypeptide encoded by an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridizable thereto, which encodes a vespid venom phospholipase, such as Polistes annularis vespid venom phospholipase A1, comprising an amino acid sequence of SEQ ID NO:64, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, wherein the immunomodulatory fragments comprise an immunomodulatory portion of a T cell epitope, or an antigenic portion of a B cell epitope of Polistes annularis phospholipase A1. Consequently, a pharmaceutical composition of the invention comprises an immunomodulatory T cell epitope of Polistes annularis venom phospholipase A1, or an antigenic portion of a B cell epitope of Polistes annularis phospholipase A1.
In another embodiment, the present invention extends to a pharmaceutical composition effective for the treatment of a vespid venom allergen-specific allergic condition comprising a polypeptide encoded by an isolated nucleic acid molecule which encodes vespid venom hyaluronidase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, and a pharmaceutically acceptable carrier thereof, wherein the polypeptide comprises an antigenic portion of a B cell epitope of vespid venom hyaluronidase, or an immunomodulatory portion of a T cell epitope of vespid venom hyaluronidase. A particular example of a pharmaceutical composition of the invention comprises a polypeptide encoded by an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:67, degenerate variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, or an isolated nucleic acid molecule hybridizable thereto, and a pharmaceutically acceptable carrier thereof, wherein the polypeptide comprises an antigenic portion of a B cell epitope of Polistes annularis hyaluronidase, or an immunomodulatory portion of a T cell epitope of Polistes annularis hyaluronidase. Examples of pharmaceutically acceptable carriers for pharmaceutical compositions of the invention are set forth infra.
Naturally, the present invention extends to a method for treating a vespid venom allergen-specific allergic condition comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention, examples of which are set forth above. Administration of a pharmaceutical composition of the invention can occur parenterally, and particularly orally. pulmonarily, nasally, topically or systemically.
Furthermore, the present invention extends to an agent for treating an immune system related disease or disorder, or a symptom of the immune system related disease or disorder, wherein the agent comprises an isolated polypeptide encoded by an isolated nucleic acid molecule which encodes a vespid venom enzyme, wherein the polypeptide comprises an immunomodulatory fragment of a vespid venom enzyme. More particularly, an agent for treating an immune system related disease or disorder, or symptom related thereto, comprises a polypeptide encoded by an isolated nucleic acid molecule which encodes a vespid venom enzyme, wherein the vespid venom enzyme comprises phospholipase, and the polypeptide comprises an immunomodulatory portion of a T cell epitope, or an antigenic portion of a B cell epitope of vespid venom phospholipase. Hence, the polypeptide of the agent can be encoded by an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the polypeptide comprises an immunomodulatory portion of a T cell epitope of Polistes annularis phospholipase A1, or an antigenic portion of a B cell epitope of Polistes annularis phospholipase A1. An agent of the invention can also comprise an isolated polypeptide encoded by an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:63, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the polypeptide comprises an immunomodulatory portion of a T cell epitope of Polistes annularis phospholipase A1, or an antigen portion of a B cell epitope of Polistes annularis phospholipase A1.
An agent for treating an immune system related disease or disorder, or a symptom related thereto, may also be derived from vespid venom hyaluronidase. Hence, an agent for treating an immune system related disorder or disease, or a symptom thereof, comprises an isolated polypeptide encoded by an isolated nucleic acid molecule which encodes a vespid venom hyaluronidase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof. Hence, an agent of the invention comprises an isolated polypeptide encoded by an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:67, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the polypeptide comprises an antigenic B cell portion Polistes annularis hyaluronidase, or an immunomodulatory portion of a T cell epitope of Polistes annularis hyaluronidase. Likewise, an agent of the invention comprises an isolated polypeptide encoded by an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:67, degenerate variants thereof, fragments thereof, or analogs or derivatives thereof, wherein the polypeptide comprises an immunomodulatory portion of a T cell epitope of Polistes annularis hyaluronidase, or an antigen portion of a B cell epitope of Polistes annularis hyaluronidase.
Furthermore, the present invention extends to a pharmaceutical composition for treating an immune system related disease or disorder, or a symptom related thereto, wherein the pharmaceutical composition comprises an agent for treating an immune system related disease or disorder, or symptom related thereto, and a pharmaceutically acceptable carrier thereof. Examples of such agents are set forth above.
Naturally, the present invention extends to a method for treating an immune system related disease or disorder, or a symptom related thereto, wherein the method comprises administering to a subject a therapeutically effective amount of a pharmaceutical composition for treating an immune system related disease or disorder, or a symptom related thereto. Examples of such pharmaceutical compositions are set forth above. Administration of a pharmaceutical composition for treating an immune system related disease or disorder to a subject can be carried out parenterally, and particularly orally, pulmonarily, nasally, topically or systemically. Furthermore, numerous diseases or disorders related to the immune system can be treated with the present invention. Examples include, but are no limited to, a pathogenic disease or disorder such as a viral disease or disorder, e.g., HIV, Herpes Simplex virus, or papiloma virus; an autoimmune disease e.g. arthritis or Lupus; or a combination of such diseases or disorders.
In addition the present invention extends to an agent for modulating immune response towards an immunogen in a mammal, wherein the agent comprises a polypeptide encoded by an isolated nucleic acid molecule which encodes a vespid venom enzyme, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof. In a particular embodiment, the present invention extends to an agent for modulating immune response towards an immunogen in a mammal as set forth above, wherein the vespid venom enzyme comprises phospholipase or hyaluronidase. Hence, an agent of the invention which modulates immune response towards an immunogen in a mammal comprises a polypeptide encoded by SEQ ID NO:63 or SEQ ID NO:67, degenerate variants thereof, fragments thereof, of analogs or derivatives thereof, wherein the polypeptide comprises an immunomodulatory portion of a T cell epitope of Polistes annularis phospholipase A1 or hyaluronidase. Moreover, the polypeptide may also comprise an antigen portion of a B cell epitope of Polistes annularis phospholipase A1 or hyaluronidase.
Moreover, the present invention extends to a pharmaceutical composition for modulating immune response of a mammal towards an immunogen, wherein the pharmaceutical composition comprises an agent of the invention for modulating immune response towards an immunogen in a mammal, as set forth above, and a pharmaceutically acceptable carrier thereof.
What""s more, the present invention extends to a method for modulating immune response in a mammal towards an immunogen, Such a method comprises administering to a mammal a therapeutically effective amount of a pharmaceutical composition of the invention for modulating immune response towards an immunogen. Examples of immunogens against which a mammal""s immune response can be modulated include a pathogen, a fragment of a pathogen, a virus, a fragment of a virus, an initiator of autoimmune disease, or a mediator of autoimmune disease. Particular examples of such viruses comprise HIV, Herpes Simplex virus, or a papiloma virus. In a particular embodiment, the modulation of the immune system results in an increased immune response towards the immunogen relative to immune response towards the immunogen prior to administration of a pharmaceutical composition of the invention.
Accordingly, it is an object of the invention to provide isolated nucleic acid molecules which encode vespid venom phospholipase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, and vespid hyaluronidase, conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof. Particular examples include, but certainly are not limited to, phospholipase A1 and hyaluronidase of Polistes annularis. It is a particular advantage of the present invention that the nucleic acid sequences encoding a number of vespid venom enzymes, in particular phospholipase and hyaluronidase, are provided. Such nucleic acid sequences allow deduction of the amino acid sequence of the vespid venom enzymes. Knowledge of the amino acid sequence allows for the determination of relevant T cell and B cell epitopes of an enzyme. More importantly, the immunodominant T cell and B cell epitopes can be determined for each enzyme allergen-sensitive individual or group of individuals, i.e., who share a susceptible MHC haplotype, or for whom the T cell epitope favors class switch events to IgE class antibodies. Once such T cell and B cell epitopes are determined, it is possible to devise immunological therapies for vespid venom enzyme-specific allergic conditions, e.g., for sensitivity to vespid venom phospholipase or hyaluronidase, or both.
It is another object of the invention to provide the DNA sequence of isolated nucleic acid molecules that encode Polistes annularis hyaluronidase, conserved variants thereof, fragments thereof, or analogs or derivatives thereof.
It is still yet another object of the invention to provide amino acid sequences of Polistes annularis phospholipase A1 and hyaluronidase, along with conserved variants thereof, fragments thereof, including immunomodulatory portions of T cell epitopes and antigenic portions of B cell epitopes of Polistes annularis phospholipase A1 and hyaluronidase. The deduced amino acid sequences of phospholipase A1 and hyaluronidase, from Pol a allow comparison of their homology to analogous enzymes from other vespids. This information provides a basis for evaluating cross-reactivity of the allergens, which can be important for allergic reactions and for therapeutic treatments. Hence, the present invention enables one of ordinary skill in the art to determine and evaluate the degree of similarity of phospholipase A1 and hyaluronidase of Pol a to environmental proteins and/or autologous proteins. It is believed that similarity of the vespid venom enzymes to such environmental proteins, and particularly to autologous proteins, has important implications for the allergic response.
It is yet still another object of the invention to provide expression and cloning vectors comprising an isolated nucleic acid molecule encoding Polistes annularis phospholipase A1 and hyaluronidase, including fragments comprising an immunomodulatory portion of a T cell epitope or an antigenic portion of a B cell epitope of these vespid venom enzymes so that the isolated nucleic acid molecules can be reproduced and expressed.
Yet another object of the invention comprises production of vespid venom enzymes such as phospholipase and hyaluronidase, along with conserved variants thereof, immunomodulatory fragments thereof, or analogs or derivatives thereof, using expression vectors of the invention.
Yet still another object of the invention is to provide agents and pharmaceutical compositions for treating an allergen-specific allergic condition in a subject, wherein the agents and pharmaceutical composition comprise an isolated polypeptide encoded by an isolated nucleic acid molecules which encodes a vespid venom enzyme, such as phospholipase or hyaluronidase, particularly from Polistes annularis, wherein the polypeptide comprises an antigen portion of a B cell epitope, or an immunomodulatory portion of a T cell epitope of Polistes annularis phospholipase A1 or hyaluronidase.
Yet still another object of the invention is to provide a method for treating a vespid venom allergen-specific allergy in a subject, wherein a pharmaceutical composition for treating an allergen-specific allergic condition is administered to the subject.
Yet still another object of the invention is to provide agents and pharmaceutical compositions comprising such agents that treat an immune system related disease or disorder in mammal, such as a pathogenic disease or disorder, a viral disease or disorder, an autoimmune disease or disorder, or a combination of immune system related diseases or disorders.
Still yet another object of the invention is to provide agents and pharmaceutical composition for modulating immune response towards an immunogen in a mammal. As a result, administration of such a pharmaceutical composition modulates the immune system""s ability to recognize and attack the immunogen. In a particular embodiment, the ability of the immune system of the mammal to recognize and attack the immunogen is increased upon administration of the pharmaceutical composition relative to the ability of the subject""s immune system to recognize and attack the immunogen prior to administration of a pharmaceutical composition of the invention.